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The L-type Ca2+ channels blocker nifedipine represses mesodermal fate determination in murine embryonic stem cells.

Nguemo F, Fleischmann BK, Gupta MK, Sarić T, Malan D, Liang H, Pfannkuche K, Bloch W, Schunkert H, Hescheler J, Reppel M - PLoS ONE (2013)

Bottom Line: This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro.This was accompanied by the inhibition of spontaneously occurring Ca(2+) transient and reduction of LTCCs current density (I(CaL)) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca(2+) channel α(1C) (Ca(V)1.2) subunits.Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurophysiology, University of Cologne, Cologne, Germany.

ABSTRACT
Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca(2+) channels of different cell types. Moreover, influx of Ca(2+) through L-type Ca(2+) channels (LTCCs) activates Ca(2+) signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca(2+) transient and reduction of LTCCs current density (I(CaL)) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca(2+) channel α(1C) (Ca(V)1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca(2+) concentration and pathways are essentially required for cardiac gene expression, differentiation and function.

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Nifedipine does not affect the generation of specific cardiac subtypes as well as the β-adrenergic and muscarinic regulation pathways.(A) Representative AP recordings from spontaneously beating ES cell-derived CMs revealed differentiation of nifedipine-treated cell into the different cardiac subtypes: nodal-, embryonic atrial- and embryonic ventricular-like CMs, note the presence of unspecified CMs (left panel). Statistic analysis (right panel) of different cardiac subtypes generated under control and nifedipine-treated cultures. (B–C) Representative AP of untreated (B) and nifedipine-treated (C) ES cells-derived CMs showed a prominent positive chronotropic effect of Iso (1 µM) (middle left) and negative chronotropic response to CCh (1 µM) (middle right) application. These effects could be partially reversed by washout. The dotted lines indicate the zero current level.
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pone-0053407-g004: Nifedipine does not affect the generation of specific cardiac subtypes as well as the β-adrenergic and muscarinic regulation pathways.(A) Representative AP recordings from spontaneously beating ES cell-derived CMs revealed differentiation of nifedipine-treated cell into the different cardiac subtypes: nodal-, embryonic atrial- and embryonic ventricular-like CMs, note the presence of unspecified CMs (left panel). Statistic analysis (right panel) of different cardiac subtypes generated under control and nifedipine-treated cultures. (B–C) Representative AP of untreated (B) and nifedipine-treated (C) ES cells-derived CMs showed a prominent positive chronotropic effect of Iso (1 µM) (middle left) and negative chronotropic response to CCh (1 µM) (middle right) application. These effects could be partially reversed by washout. The dotted lines indicate the zero current level.

Mentions: To test whether loss of gene expression actually reflects CMs with distinct functional properties, we performed patch-clamp measurement to record spontaneous action potentials (APs). In control CMs (n = 32) and in CMs derived from nifedipine-treated group (n = 21) we could measured APs of all 3 major cardiomyocytes subtypes. These could be classified as nodal-like (9.3% vs. 9.5%), embryonic atrial-like (12.50% vs. 14.2%) and ventricular-like (78.1% vs. 76.2%) APs (Figure 4A, left), respectively based on their shape and properties as described previously [19], [34] and summarized in Table 1. However, some CMs in the control (n = 14) and nifedipine-treated (n = 9) groups did not match any of the cardiac subtype and were classified as unspecified. The percentages of different AP subtypes recorded in CMs derived from the control and nifedipine treatment group were similar (Figure 4A, right). The presence of nifedipine during the differentiation process led to a significant alteration of AP parameters such as beating frequency, amplitude and AP duration at 90% of repolarization (Table 1).


The L-type Ca2+ channels blocker nifedipine represses mesodermal fate determination in murine embryonic stem cells.

Nguemo F, Fleischmann BK, Gupta MK, Sarić T, Malan D, Liang H, Pfannkuche K, Bloch W, Schunkert H, Hescheler J, Reppel M - PLoS ONE (2013)

Nifedipine does not affect the generation of specific cardiac subtypes as well as the β-adrenergic and muscarinic regulation pathways.(A) Representative AP recordings from spontaneously beating ES cell-derived CMs revealed differentiation of nifedipine-treated cell into the different cardiac subtypes: nodal-, embryonic atrial- and embryonic ventricular-like CMs, note the presence of unspecified CMs (left panel). Statistic analysis (right panel) of different cardiac subtypes generated under control and nifedipine-treated cultures. (B–C) Representative AP of untreated (B) and nifedipine-treated (C) ES cells-derived CMs showed a prominent positive chronotropic effect of Iso (1 µM) (middle left) and negative chronotropic response to CCh (1 µM) (middle right) application. These effects could be partially reversed by washout. The dotted lines indicate the zero current level.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3539992&req=5

pone-0053407-g004: Nifedipine does not affect the generation of specific cardiac subtypes as well as the β-adrenergic and muscarinic regulation pathways.(A) Representative AP recordings from spontaneously beating ES cell-derived CMs revealed differentiation of nifedipine-treated cell into the different cardiac subtypes: nodal-, embryonic atrial- and embryonic ventricular-like CMs, note the presence of unspecified CMs (left panel). Statistic analysis (right panel) of different cardiac subtypes generated under control and nifedipine-treated cultures. (B–C) Representative AP of untreated (B) and nifedipine-treated (C) ES cells-derived CMs showed a prominent positive chronotropic effect of Iso (1 µM) (middle left) and negative chronotropic response to CCh (1 µM) (middle right) application. These effects could be partially reversed by washout. The dotted lines indicate the zero current level.
Mentions: To test whether loss of gene expression actually reflects CMs with distinct functional properties, we performed patch-clamp measurement to record spontaneous action potentials (APs). In control CMs (n = 32) and in CMs derived from nifedipine-treated group (n = 21) we could measured APs of all 3 major cardiomyocytes subtypes. These could be classified as nodal-like (9.3% vs. 9.5%), embryonic atrial-like (12.50% vs. 14.2%) and ventricular-like (78.1% vs. 76.2%) APs (Figure 4A, left), respectively based on their shape and properties as described previously [19], [34] and summarized in Table 1. However, some CMs in the control (n = 14) and nifedipine-treated (n = 9) groups did not match any of the cardiac subtype and were classified as unspecified. The percentages of different AP subtypes recorded in CMs derived from the control and nifedipine treatment group were similar (Figure 4A, right). The presence of nifedipine during the differentiation process led to a significant alteration of AP parameters such as beating frequency, amplitude and AP duration at 90% of repolarization (Table 1).

Bottom Line: This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro.This was accompanied by the inhibition of spontaneously occurring Ca(2+) transient and reduction of LTCCs current density (I(CaL)) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca(2+) channel α(1C) (Ca(V)1.2) subunits.Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurophysiology, University of Cologne, Cologne, Germany.

ABSTRACT
Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca(2+) channels of different cell types. Moreover, influx of Ca(2+) through L-type Ca(2+) channels (LTCCs) activates Ca(2+) signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca(2+) transient and reduction of LTCCs current density (I(CaL)) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca(2+) channel α(1C) (Ca(V)1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca(2+) concentration and pathways are essentially required for cardiac gene expression, differentiation and function.

Show MeSH